Compositions of somatostatin analogues

ABSTRACT

In one embodiment, a composition of somatostatin analogues having the general formula: 
                         
is provided wherein Z may be absent or present and when present is selected from DOTA- and DTPA-based chelators, NOTA-based chelators, carbonyl compounds, hydrazino nicotinamide, N 4 -chelators, desferrioxamine, N x S y -chelators, optionally complexed or labeled with a radioisotope, tyrosine for halogenation, a fluorescent dye, or biotin. The composition further provides that L may or may not be present and when present is a linker molecule, X1 is glutamic acid or a symmetric or asymmetric diamino acid containing 3 or 4 consecutive C atoms, X2 is a positively charged natural or unnatural amino acid, an arginine mimic, citrulline, or a neutral amino acid, X3 is phenylalanine, Ala-[3-(2-thienyl)], α-naphthylalanine, or β-naphthylalanine, X4 is an aromatic amino acid, X5 is threonine or serine, and X6 is phenylalanine, Ala-[3-(2-thienyl)], α-naphthylalanine, or β-naphthylalanine.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to somatostatin analogues and their use indiagnosis and therapy. The invention also relates to pharmaceuticalcompositions comprising the novel analogues.

2. Description of the Related Art

Somatostatin (somatotroph release-inhibiting factor), was initiallydiscovered as a hypothalamic neurohormone that inhibits growth hormonesecretion. It is a widely distributed peptide in both the central andperipheral nervous system and is also present in peripheral tissuesincluding the endocrine pancreas, gut, thyroid, adrenals and kidneys. Inaddition, somatostatin is produced by inflammatory and immune cells aswell as many cancer cells.

In mammals, two forms of bioactive peptides, somatostatin 14 andsomatostatin 28 are found. They are produced by tissue-specificproteolytic processing of a common precursor. The natural somatostatinpeptides have a short half-life, which is why many somatostatinanalogues have been synthesized. Among them, octreotide, lanreotide andvapreotide have been intensively investigated and are in clinical usefor the medical treatment of acromegaly and neuroendocrine tumors. Theseoctapeptides retain the amino acid residues (or substitutes) within acyclic peptide backbone that are involved in the biological effect ofthe peptide (Phe⁷ or Tyr⁷, D-Trp⁸, Lys⁹ and Thr¹⁰ or Val¹⁰) and displaymarkedly increased stability.

The biological effects of somatostatin are mediated by specific plasmamembrane receptors that have been identified in normal and neoplastictissues by binding studies and receptor autoradiography techniques. Fivesomatostatin receptor genes have been cloned from human and mammalianlibraries and designated sst1 to sst5 receptors. The sst subtypes belongto the family of G protein-coupled receptors with seventransmembrane-spanning domains and present a high degree of sequenceidentity (39–57%). The sequence differences reside in the extracellularand intracellular domains and are probably responsible for theirsignalling specificity.

All somatostatin receptors bind somatostatin 14 and somatostatin 28 witha high affinity (nM range), although with a slightly higher affinity forsomatostatin 14. However, the receptors show major differences in theiraffinities for peptide analogues. Analogues that are known to dateexhibit a low affinity for sst1 and sst4 whereas they bind the sst2 andsst5 receptor with a high affinity, comparable to that of somatostatin14 and bind the sst3 receptor with moderate affinity.

In addition to its effect on secretion and intestinal motility,somatostatin inhibits the proliferation of normal as well as tumorcells. The antiproliferative action of somatostatin can be signalled viathe five sst receptors which initiate pertussis toxin-sensitive Gprotein-dependent cell growth arrest or apoptosis according-to receptorsubtypes and target cells.

When expressed in CHO cells, ligand-activated sst1, sst2A, sst4, andsst5 receptors inhibit mitogenic signal of serum or growth factors as aresult of hypophosphorylation of the retinoblastoma gene product (Rb)and G₁ cell cycle arrest.

However, distinct signal transduction pathways are involved in thesomatostatin-induced G₁ cell cycle arrest depending on receptor subtype.The sst1 receptor mediates cell growth arrest through the stimulation ofthe tyrosine phosphatase SHP-2, activation of the Ras/MAP kinase ERKpathway and induction of the cyclin-dependent kinase inhibitorp21^(waf1/Cip1), whereas the sst5 receptor acts by a mechanism involvinga dephosphorylation cascade leading to inhibition of guanylate cyclase,cGMP-dependent protein kinase G and MAP kinase ERK 1/2.

The antiproliferative effect mediated by the sst2 receptor results fromthe activation of the tyrosine phosphatase SHP-1 and thedephosphorylation of activated growth factor receptors thus leading tothe negative regulation of growth factor-induced mitogenic signalling.

In addition, somatostatin-activated SHP-1 induces a G₁ cell cyclearrest, upregulates the cyclin-dependent kinase inhibitor p^(27Kip1)leading to the accumulation of hypophosphorylated Rb.

The antiproliferative effect of somatostatin can also result fromapoptosis. Apoptosis is induced by sst3 as a result of the induction ofp53 and Bax. In human pancreatic cancer cells expressing mutated p53 anddevoid of endogenous sst2 receptor, correction of the deficit byexpression of sst2 receptor induces an increase in cell death indicatingthat somatostatin can induce apoptosis by p53-dependent andp53-independent mechanisms.

The antiproliferative effects of somatostatin result from its actionsvia the endocrine pathway, but evidence exists that somatostatin canalso act via an autocrine/paracrine pathway. Immunoreactive somatostatinhas been found in somatostatin receptor-positive normal and tumor celltypes such as endocrine, lymphoid cells, macrophages, breast cancercells, colonic tumor cell and additionally, somatostatin mRNA isdetected in a wide variety of neuroendocrine tumors known to expresssomatostatin receptors. Correction of the sst2 receptor deficit in humanpancreatic cancer cells by sst2 receptor expression induces a negativeautocrine loop in the absence of exogenous ligand, which is due to sst2receptor-induced expression and secretion of endogenous sst2 ligand(somatostatin 14 and somatostatin 28). This results in inhibition ofcancer cell proliferation and reversion of cell tumorigenicity in vitroand in vivo after xenografts in nude mice.

The somatostatin effect on tumor growth may be the result of indirecteffects of the peptide resulting from the inhibition of secretion ofgrowth-promoting hormones and growth factors which specifically regulatetumor growth. For example, the secretion of insulin-like growth factor-1(IGF-1) which is produced by hepatocytes through GH-dependent and-independent mechanisms is negatively controlled by octreotide as aresult of an effect on GH secretion and the sst2 and sst5 receptors havebeen demonstrated to be implicated in this effect. In addition,somatostatin can decrease IGF gene expression. Somatostatin alsoinhibits angiogenesis. Overexpression of peritumoral vascularsomatostatin receptors with high affinity for somatostatin andoctreotide has been reported in human peritumoral colorectal carcinomas,small cell lung carcinoma, breast cancer, renal cell carcinoma andmalignant lymphoma. This expression appears to be independent ofreceptor expression in the tumor. It may reflect the presence of sstreceptors in the venous smooth muscle cells as well as endothelial cellsand may allow a vasoconstriction resulting in local hypoxia of the tumoror inhibition of endothelial cell growth and monocyte migration. Sst2,sst3 or sst5 receptors might be involved in these effects.

Although the biological role and cellular distribution of each receptorsubtype are not yet completely understood it is clear that thedevelopment of analogues binding to all somatostatin receptor subtypes,so-called pansomatostatin, has high potential.

In one embodiment herein, it is thus an object of the present inventionto provide such new somatostatin analogues that bind to all fivesomatostatin receptors and have a higher half-life (high metabolicstability) than somatostatin itself.

DETAILED DESCRIPTION

This is achieved according to the invention by somatostatin analogues ofthe general formula:

wherein:

-   Z may be absent or present and when present is selected from the    group consisting of DOTA- and DTPA-based chelators, NOTA-based    chelators, carbonyl compounds, 2hydrazino nicotinamide (hynic),    N₄-chelators, desferrioxamin, N_(x)S_(y)-chelators, all optionally    complexed with a radioisotope, Tyrosine (Tyr) for halogenation, a    fluorescent dye or biotin;-   L may or may not be present and is a linker molecule;-   X1 is a symmetric or asymmetric diamino acid, containing 3 or 4    consecutive C atoms with a linker to the chelating agent, for    example D/L-diamino butyric acid (D/L-Dab) for a more basic    character or D/L-Glu for coupling to primary and secondary amino    groups;-   X2 is a positively charged natural or unnatural amino acid or    arginine mimic or citrulline, or a neutral amino-acid like Asn;-   X3 is phenylalanine (Phe), Ala-[3-(2-thienyl)] or    α-,β-naphthylalanine;-   X4 is an aromatic amino acid, optionally halogenated, in particular    with Cl, Br, I or ¹⁸F;-   X5 is threonine (Thr) or serine (Ser); and-   X6 is phenylalanine (Phe), Ala-[3-(2-thienyl)] or    α-,β-naphthylalanine.

X1 is preferably diamino propionic acid or diamino butyric acid.

Preferably X2 is selected from the group consisting of Lysine (Lys),homolysine,

ornithine and

L/D-Phg(4-amidino) and derivatives.When X2 is an arginine mimic it is preferably a group selected from:

(R/S)-Gly/Ala-4-Pip(N-amidino)

D/L-Phe[4-guanidino] and derivatives thereof

and quatemary ammonium derivatives (NR₄ ⁺).

X4 is preferably selected from the group consisting of tyrosine (Tyr),halogenated tyrosine, in particular iodinated tyrosine (I-Tyr),dimethyltyrosine (diMe-Tyr), α-,β-Naphthylalanine, halogenatedphenylalanine, in particular iodated phenylalanine (I-Phe). When X4 ishalogenated this is preferably with Cl, Br or I.

When a linker L is present, it may be selected from the group consistingof tyrosine, lysine, diaminobutyric acid, diaminopropionic acid,polyethylene glycol, fatty acids and their derivatives, β-alanine,5-amino valeric acid, sarcosine, gluceronic acid. Alternatively, thelinker function may be taken on by X1. An example of a symmetric linkeris

diamino acids, like diaminobutyric acid or 4,8 diaminooctanoic acid andsymmetric molecules, like N,N-bis-(N′-3-aminopropyl)glycine.

In case Z is complexed with a radioisotope, the isotope may be selectedfrom the group consisting of ⁶⁷Ga, ⁶⁸Ga, ^(103m)Rh, ^(195m)Pt,^(114m)In, ¹⁸⁶Re, ¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁶Ga, ⁶⁷Cu, ¹⁶⁹Er, ^(117m)Sn, ¹²¹Sn,¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁴⁹Tb, ¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴⁹Pm,¹⁵¹Pm, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷²Tm, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh,¹¹¹Ag, ²¹³Bi, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I and ²¹¹At.

Preferred embodiments of the invention are somatostatin analogues offormula I wherein X2 is arginine, or wherein X3 is phenylalanine, orwherein X4 is phenylalanine, or wherein X4 is tyrosine, or wherein X5 isthreonine, or wherein X6 is phenylalanine or wherein X1 isdiaminobutyric acid.

The invention further relates to analogues wherein one or more of theabove substituents are combined. In such analogues, the groups that donot have one of the above substituents are as defined for the generalformula I above.

When all the above substituents are combined an analogue results havingthe general formula:

wherein Z may or may not be present and is as defined above. Inpreferred embodiments of the invention Z is DOTA, DOTAGA or tyrosine.

When X1 is D/L-Glu L are amine ending molecules like D/L-Lys, and Z arechelating agents likep-NH₂-Bz-DOTA(2-p-aminobenzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid), DOTA-p-NH₂-anilide(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acidmono(p-aminoanilide) and MeO—NH₂-Ph-DOTA(5-amino-2-methoxyphenyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid).

Z may be complexed with a radioisotope, or coupled to a fluorescent dyeor biotin. For use in diagnosis the analogue may be labeled with aradioactive metal isotope selected from the group consisting of^(99m)Tc, ²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ¹¹¹In, ^(113m)In, ¹²³I, ¹⁷⁷Lu, ⁹⁷Ru,⁶²Cu, ⁶⁴Cu, ⁵²Fe, ^(52m)Mn, ⁵¹Cr, ¹²⁴I and ¹⁸F.

Suitable fluorescent dyes are cyanin-dyews. Such dyes may be used whenthe analogues are applied in in vitro and in vivo diagnosis. Biotin isuseful as a label in histology.

The somatostatin analogues of the invention can be used as a medicamentin the treatment of diseases that are characterized by an overexpressionof one or more somatostatin receptors, in particular the treatment maybe directed to tumors bearing one or more somatostatin receptors.Examples of such tumours are neuroendocrinic tumors, astrocytoma, lungcancer, lymphoma, mama carcinoma, pancreatic tumors, thyroid cancer,colon cancer, SCLC, renal cell cancer.

For therapy, the somatostatin analogues of the invention may be labeledwith a radioisotope selected from the group consisting of ^(114m)In,¹⁸⁶Re, ¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁶Ga, ⁶⁷Cu, ¹⁶⁹Er, ^(117m)Sn, ¹²¹Sn, ¹²⁷Te,¹⁴²Pr, ¹⁴³Pr, ^(195m)Pt, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁴⁹Tb, ¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁵Dy,¹⁴⁹Pm, ¹⁵¹Pm, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷²Tm, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu,¹⁰⁵Rh, ^(103m)Rh, ¹¹¹Ag, ¹²⁴I, ¹³¹I and ²¹¹At.

The invention also relates to the use of the somatostatin analogues forthe preparation of a pharmaceutical composition for treatment ordiagnosis.

In case the pharmaceutical composition is a diagnostic composition, thesomatostatin analogue(s) is(are) labeled with a radioactive metalisotope selected from the group consisting of ^(99m)Tc, ²⁰³Pb, ⁶⁷Ga,⁶⁸Ga, ⁷²As, ¹¹¹In, ^(113m)In, ¹²³I, ¹⁷⁷Lu, ⁹⁷Ru, ⁶²Cu, ⁶⁴Cu, ⁵²Fe,^(52m)Mn and ⁵¹Cr. When the pharmaceutical composition is atherapeutical composition, the somatostatin analogue(s) is(are) labeledwith a radioisotope selected from the group consisting of ^(114m)In,¹⁸⁶Re, ¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁶Ga, ⁶⁷Cu, ¹⁶⁹Er, ^(117m)Sn, ¹²¹Sn, ¹²⁷Te,¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁴⁹Tb, ¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴⁹Pm, ¹⁵¹Pm,¹⁵³Sm, ¹⁵⁷Gd, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷²Tm, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh,^(103m)Rh, ^(195m)Pt, ¹¹¹Ag, ¹²⁴I, ¹³¹I and ²¹¹At.

The invention furthermore relates to a pharmaceutical compositioncomprising a suitable excipient and one or more of the somatostatinanalogues.

The present invention will be further elucidated in the followingexamples that are given for illustration purposes only and are in no wayintended to limit the invention.

EXAMPLES Example 1 General Method for the Synthesis ofChelator-Somatostatin Analogues of the Invention

The synthesis to the linear peptide intermediates is performed withwell-established solid phase methods with Fmoc as transient aminoportecting group, and TFA-labile side chain protection groups as Pbf forArg, BOC for Trp and Lys, tBu for Thr. For a description of the methodsee “Fluorenylmehoxycarbonylpolyamide solid phase synthesis—A practicalapproach” by E. Atherton and R. C. Sheppard, Information Press Ltd.,Oxford, England (1989). The Solid phase peptide synthesis is carried outon a semiautomatic peptide synthesiser.

The diaminobutyric acid is used with Fmoc as γ-amino protection group Zas α-amino protecting group. Coupling reactions are done with in-situprepared hydroxy-benzotriazole esters with DIC, but any other couplingreagent may be used, e.g. the well introduced isourea derivatives (HATUetc.). The first amino acid (Fmoc-Phe-OH) is esterified to a superacid-labile linker (trialkoxy-benzhydryl or chloro-trityl) on the resin.After synthesis, the peptides are routinely cleaved mildly from theresin, using 20% acetic acid in dichloromethane. Coevaporation withtoluene is performed two times leaving intact side-chain protectiongroups and therefore allowing subsequently cyclisation via carboxamideformation, using 10 equivalents ofdicyclohexylcarbodiimide/hydroxybenzotrialoze in DMF at high dilution.

The crude product was extracted three times between ethyl acetate and a5% aqueous oxalic solution and the organic layer was concentrated todryness. The Z-protecting group is then selectively removed by catalytichydrogenolysis, using Pd/C as catalyst in methanol, without significantaffection of the indole system. The products were filtered and purifiedwith a SepPak cartridge C₁₈ (Macherey-Nagel, Düren, Germany) using waterand methanol as eluents. The resulting free amino acid group serves asan attaching point for a carboxylic functionalised chelator derivative,which may include an appropriate spacer. After the coupling to aprochelator, the peptide conjugate is deprotected with a solution oftrifluoroacetic acid/phenole/thioanisol/water 85:5:5:5 for 2–5 hours.The final product was precipitated in isopropylether/petrolether 1:1 andpurified by C₁₈ reverse phase chromatography (Metrohm LC CaDi 22-14,column: Macherey-Nagel, Düren, Germany) with a purity >98%.

Example 2 Synthesis ofγ-9-fluorenylmetbyloxycarbonyl-α-benzyl-oxycarbonyl-D-diaminobuc acid(Z-Dab(Fmoc)-OH)

α-Benzyloxycarbonyl-D-diaminobutyric acid (Z-Dab-OH) is commerciallyavailable (Bachem, Bubendorf, Switzerland). This starting material wasdissolved in acetone/water 1:1, sodium carbonate was added to a final pHof 9–10 and treated with9-fluorenylmethyloxycarbonyl-N-hydroxysuccinimide (Fmoc-OSu). Thereaction mixture was stirred for 18 hours at RT, cooled to 5° C.;ethylacetate was added and then acidified with 6 N HCl. The organicphase was washed with water four times, dried over sodium sulfate andconcentrated. The product was recrystallised fromethylacetate/petrolether.

Example 3 Chelator Coupling

Three equivalents of prochelator (for example DOTAGA(tBU)₄(1-(1-carboxy-3-carbotertbutoxypropyl)-4,7,10-(carbotertbutoxymethyl)-1,4,7,10-tetraazacyclododecane),DOTA(tBu)₃(1,4,7-tris(carbotertbutoxymethyl)-10-carboxymethyl-1,4,7,10-tetraazacyclododecane),DTPA(tBu)₄(1,1,7,7-tetrakis(carbotertbutoxymethyl)-4-carboxy-1,4,7-triazapentane)were incubated with 3 equivalents of HATU(O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphatein DMF (dimethylformamide) for 30 min. The Z deprotected diamino acids(see above) were treated with this solution for 4 h at RT.

After concentration the mixture was dissolved in ethylacetate andextracted three times between 5% sodium hydrogencarbonate solution andethylacetate. The organic layer was evaporated to dryness and the crudeproduct is deprotected (see above).

A typical labeling procedure with ¹¹¹In and ⁹⁰Y is as follows.“PanSomatostatin” is one of the somatostatin analogues of the invention.

A buffer solution is prepared by dissolving 328 mg sodium acetate and370 mg gentisic acid in 10 ml of water suprapure and adjusting the pH topH 5 with 1 M sodium hydroxide.

10 μg of peptide (DOTA-PanSomatostatin), 100 μl of buffer and 120 μl ofYttrium-90 chloride (2.9 GBq/ml 0.05M HCl) were incubated at 95° C. for40 min, cooled to room temperature and the labeling yield andradiochemical purity was determined by RP-HPLC using a Macherey-NagelNucleosil-C₁₈ column and a linear gradient from 5% acetonitrile/0.1%trifluoroacetic acid to 60% acetonitrile/0.1% trifluoroacetic acid in 30min. The labeling yield is >98%.

10 μg of peptide (DOTA-PanSomatostatin), 100 μl of buffer and 100 μl ofIndium-111 chloride (370 MBq/ml 0.05M HCl) were incubated at 95° C. for40 min, cooled to room temperature and the labeling yield andradiochemical purity was determined by RP-HPLC using a Macherey-NagelNucleosil-C₁₈ column and a linear gradient from 5% acetonitrile/0.1%trifluoroacetic acid to 60% acetonitrile/0.1% trifluoroacetic acid in 30min. The labeling yield is >98%. The stability is >87% after one week in10000 times excess of DTPA and 76% in freshly prepared serum after oneweek, checked by HPLC. The serum proteins were precipitated withmethanol, centrifuged and the methanol phase was injected, using thesame gradient as above.

10 μg of peptide (DOTAGA-PanSomatostatin), 100 μl of buffer and 20 μofYttrium-90 chloride (1.8 GBq/ml 0.05M HCl) were incubated at 95° C. for40 min, cooled to room temperature and the labeling yield andradiochemical purity was determined by RP-HPLC using a Macherey-NagelNucleosil-C₁₈ column and a linear gradient from 5% acetonitrile/0.1%trifluoroacetic acid to 60% acetonitrile/0.1% trifluoroacetic acid in 30min. The labeling yield is >98%. The radiochemical purity is >97% after72 h. The stability is >87% after 72 h and >74% after one week in 10000times excess of DTPA. Heating to 95° C. for 45 min did not impair theradiopeptide.

Example 4 Analysis of Affinity Profiles of Various Analogues forSomatostatin Receptors sst1–sst5

Cell culture CHO-K1 cells stably expressing human sst1 and sst5 werekindly provided by Drs. T. Reisine and G. Singh (University ofPennsylvania, Philadelphia, Pa.) and CCL39 cells stably expressing humansst2, sst3, and sst4 by Dr. D. Hoyer (Novartis Pharma, Basel,Switzerland).

CHO-K1 cells were grown in Ham's F-12 medium and CCL39 cells inDulbecco's modified Eagle's medium/Ham's F-12 (1:1) mix, supplementedwith 10% foetal bovine serum, 100 U/ml penicillin and 100 μg/mlstreptomycin, in humidified air containing 5% CO₂ at 37° C. Geneticin(G418-sulfate; Gibco, USA) was used where necessary to maintainselection pressure at a final concentration of 400 μg/ml for sst2 tosst4—and 285 μg/ml for sst5—expressing cells as described previously[Rens-Domiano S & Reisine T. Biochemical and functional properties ofsomatostatin receptors. J. Neurochem. 58:1987–1996 (1992); OCaroll A etal., Characterization of cloned human somatostatin receptor SSTR5.Molec. Pharmacol. 46:291–298 (1994); Siehler S et al., [¹²⁵I]Tyr¹⁰-cortistatin₁₄ labels all five somatostatin receptors.Naunyn-Schmiedeberg's Arch. Pharmacol. 357:483–489 (1998)].

All culture reagents were supplied by Gibco BRL, Life Technologies,Grand Island, N.Y.

Example 5 In situ Hybridisation Histochemistry

To control adequacy of the cell material, in situ hybridisation forhuman sst mRNAs was performed on CHO-K1 and CCL39 cells expressing thedifferent sst receptor subtypes.

Cells were detached from culture flasks by washing with Puck's Saline Aand brief incubation with trypsin (0.5 mg/ml)/EDTA (0.2 mg/ml),collected by centrifugation, and resuspended in phosphate-bufferedsaline at a final cell density of approximately 6×10⁴ cells/μl. 25 μlAliquots of cell suspension were spotted onto microscopic slides, airdried, and stored at −20° C.

They were subsequently fixed with 4% formaldehyde, washed withphosphate-buffered saline, air dried, and stored at 4° C. under dryconditions. Cell smears were then used for sst1, sst2, sst3, sst4, andsst5 mRNA detection by in situ hybridisation. The protocol followed wasessentially that described in detail previously [Reubi J C et al.,Expression and localization of somatostatin receptor SSTR1, SSTR2 andSSTR3 mRNAs in primary human tumors using in situ hybridization. CancerRes. 54:3455–3459 (1994)].

Oligonucleotide probes complementary to the sst1, sst2, sst3 [Reubi1994, supra], sst4 and sst5 [Thoss VS et al., Expression of fivesomatostatin receptor mRNAs in the human brain and pituitary.Naunyn-Schmiedeberg's Arch. Pharmacol. 354:411–419 (1996)] mRNAs weresynthesised and purified on a 20% polyacrylamide—8M urea sequencing gel(Microsynth, Balgach, Switzerland). They were labelled at the 3′-end byusing [α³²P]dATP (>3000 Ci/mmol; NEN Life Science Products, Boston,Mass.) and terminal deoxynucleotidyl-transferase (Boehringer, Mannheim,Germany) to specific activities of 33.3–74 GBq/mmol. Control experimentswere carried out with the probes used in the present study to determinethe specificity of the hybridisation signal obtained, as describedpreviously [Reubi 1994, supra].

These control in situ hybridisation studies confirmed that the five celllines used for the study expressed the correct sst mRNA.

Example 6 Receptor Autoradiograhy

Cells were washed twice with and scraped into ice-cold 0.05 M Tris-HCl(pH 7.4), collected by centrifugation, and homogenised using arotor/stator slash system (Polytron, Kinematica Inc., Littau,Switzerland) in the same buffer. After centrifugation at 120 g for 5 minat 4° C., the supernatant was collected and centrifuged again at 48,000g for 30 min at 4° C. The resulting pellet was resuspended in ice-coldTris buffer, transferred into a microfuge tube, and centrifuged at20,000 g for 15 min at 4° C. After withdrawal of the supernatant, themembrane pellet was stored at −80° C.

Receptor autoradiography was performed on 20 μm thick cryostat (Leitz1720, Rockleigh, N.J.) sections of the membrane pellets, mounted onmicroscope slides, and then stored at −20° C. For each of the testedcompounds, complete displacement experiments with the universalsomatostatin radioligand ¹²⁵I-[Leu⁸, D-Trp²², Tyr²⁵]-somatostatin 28using increasing concentrations of the unlabelled peptide ranging from0.1–1000 nM were performed. The unlabelled, universal somatostatin 28was run in parallel using the same increasing concentrations, ascontrol.

IC₅₀ values were calculated after quantification of the data using acomputer-assisted image processing system as described previously [ReubiJ C et al., Detection of somatostatin receptors in surgical andpercutaneous needle biopsy samples of carcinoids and islet cellcarcinomas. Cancer Res. 50: 5969–5977 (1990)]. Tissue standards(Autoradiographic [¹²⁵I] microscales, Amersham) that contain knownamounts of isotope, cross-calibrated to tissue-equivalent ligandconcentrations were used for quantification [Reubi J C. In vitroidentification of vasoactive intestinal peptide receptors in humantumors: Implications for tumor imaging. J. Nucl. Med. 36: 1846–1853(1995)]. Advantages of the present method using receptor autoradiographywith sectioned cell pellets compared to binding on cell homogenates are,in addition to an economy on cells and a great flexibility, the greaterinter-assay reliability and reproducibility, since the same embeddedpellet can be used for successive experiments. As a minor disadvantage,IC₅₀ values are somewhat higher than in the homogenate binding assay.

The results are given in the following table. The compound tested is A:

wherein Z is varied.

Z hsst1 hsst2 hsst3 hsst4 hsst5 ss28¹⁾ — 2.9 1.8 4.6 3.0 2.3 ss28 — 5.21.4 2.7 3.9 2.4 ss28 — 5.2 2.8 3.8 5.7 3.8 A DOTA 7.6 3.0 1.6 0.9 0.8 AY-DOTA 3.5 3.7 0.9 2.1 3.2 A Y-DOTA 2.4 4.6 1.8 1.1 1.9 A Y-DOTA 4.6 133.0 2.0 1.0 A Ga-DOTA 6.0 5.2 3.0 4.1 1.0 A DOTAGA 40 3.8 0.8 3.0 1.5 ADOTAGA 24 3.8 2.5 2.3 1.9 A Y-DOTAGA 64 7.5 1.0 8.0 3.1 A Y-DOTAGA 345.0 2.6 4.4 2.7 A Tyr 1.8 0.6 1.4 1.3 0.48 A with NH₂ 24.5 2.9 2.0 1.00.8 X4 is Tyr A with — 10.5 1.8 1.7 0.68 0.57 X4 is Tyr and X1 is GABAminisoma — 172 2.8 3.2 15.3 6.8 tostatin²⁾ Arg- 20.0 4.0 2.5 10.6 2.7derivative³⁾Control substances:

wherein ab is aminobutyric acidIt is preferred according to the invention that the IC₅₀ values rangefrom 1 to 10 nM. From the above table it follows that the compounds ofthe invention show a IC₅₀ falling within the claimed range for most orall of the receptor subtypes.

1. A composition of somatostatin analogues having the general formula:

wherein: Z may be absent or present and when present is selected fromthe group consisting of DOTA-based chelators, DTPA-based chelators,NOTA-based chelators, carbonyl compounds, hydrazino nicotinamide,N₄-chelators, desferrioxamine, N_(X)S_(Y)-chelators, tyrosine forhalogenation, a fluorescent dye and biotin; L may or may not be presentand is a linker molecule; X1 is a symmetric or asymmetric diamino acidcontaining 3 or 4 consecutive C atoms; X2 is a positively chargednatural or unnatural amino acid, an arginine mimic, citrulline, or aneutral amino acid; X3 is phenylalanine, Ala-[3-(2-thienyl)],α-naphthylalanine, or β-naphthylalanine; X4 is an aromatic amino acid;X5 is threonine or serine; and X6 is phenylalanine, Ala-[3-(2-thienyl)],α-naphthylalanine or β-naphthylalanine.
 2. The composition of claim 1,wherein X2 is selected from the group consisting of diamino propionicacid and diamino butyric acid.
 3. The composition of claim 1, wherein X2is selected from the group consisting of lysine, I-Amp, ornithine, andL/D-Phg(4-amino).
 4. The composition of claim 1, wherein X2 is thearginine mimic.
 5. The composition of claim 1, wherein X4 is selectedfrom the group consisting of tyrosine, halogenated tyrosine, iodinatedtyrosine, dimethyltyrosine, α-naphthylalanine, β-naphthylalanine, andhalogenated phenylalanine.
 6. The composition of claim 1, wherein L isselected from the group consisting of tyrosine, lysine, β-alanine,sarcosine, succinic acid, and glutaric acid.
 7. The composition of claim1, wherein Z is complexed with a radioisotope selected from the groupconsisting of ^(114m)In, ¹⁸⁶Re, ¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁶Ga, ⁶⁷Cu, ¹⁶⁹Er,^(117m)Sn, ¹²¹Sn, ¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ^(195m)Pt, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁴⁹Tb,¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴⁹Pm, ¹⁵¹Pm, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷²Tm,¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh, ¹¹¹Ag, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ²⁰³Bi, ²¹¹At,and ^(103m)Rh.
 8. The composition of claim 1, wherein X2 is arginine. 9.The composition of claim 1, wherein X3 is phenylalanine.
 10. Thecomposition of claim 1, wherein X4 is phenylalanine.
 11. The compositionof claim 1, wherein X4 is tyrosine.
 12. The composition of claim 1,wherein X5 is threonine.
 13. The composition of claim 1, wherein X6 isphenylalanine.
 14. The composition of claim 1, wherein X1 is diaminobutyric acid.
 15. The composition of claim 1 having the general formula:


16. The composition of claim 1, wherein Z is DOTA.
 17. The compositionof claim 1, wherein Z is DOTAGA.
 18. The composition of claim 1, whereinZ is tyrosine.
 19. The composition of claim 1, wherein L is absent. 20.The composition of claim 1, further comprising a radioactive isotopeselected from the group consisting of ^(99m)Tc, ²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁷²As,¹¹¹In, ¹¹³In, ¹²³I, ¹⁷⁷Lu, ⁹⁷Ru, ⁶²Cu, ⁶⁴Cu, ⁵²Fe, ^(52m)Mn, ⁵¹Cr, ¹²⁴Iand ¹⁸F.
 21. The composition of claim 1, further contained within apharmaceutical composition comprising a suitable excipient.
 22. Thecomposition of claim 21, further comprising a radioactive isotopeselected from the group consisting of ^(99m)Tc, ²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁷²As,¹¹¹In, ^(113m)In, ¹²³I, ¹²⁴I, ¹⁸F, ¹⁷⁷Lu, ⁹⁷Ru, ⁶²Cu, ⁶⁴Cu, ⁵²Fe,^(52m)Mn and ⁵¹Cr.
 23. The composition of claim 21, further comprising aradioisotope selected from the group consisting of ^(114m)In, ¹⁸⁶Re,¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁶Ga, ⁶⁷Cu, ¹⁶⁹Er, ^(117m)Sn, ¹²¹Sn, ¹²⁷Te, ¹⁴²Pr,¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁴⁹Tb, ¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴⁹Pm, ¹⁵¹Pm, ¹⁵³Sm,¹⁵⁷Gd, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷²Tm, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh, ¹¹¹Ag, ¹²⁴I and¹³¹I.
 24. The composition of claim 1, wherein X1 is D/L-diamino butyricacid.
 25. The composition of claim 1, wherein X1 is D/L-diaminopropionic acid.
 26. The composition of claim 1, wherein Z is a chelatingagent selected from the group consisting of p-NH₂-Bz-DOTA,DOTA-p-NH₂-anilide, and MeO—NH₂-Ph-DOTA.
 27. The composition of claim 1,wherein X2 is asparagine.
 28. The composition of claim 1, wherein X4 isa halogenated aromatic amino acid.
 29. The composition of claim 4,wherein the arginine mimic is selected from the group consisting ofR/S-Gly-Ala-4-Pip(N-amidino), D/L-Phe(4-guanidino), andR/S-2-amino-4-[4-(2-amino)pyrimidinyl]butanoic acid.
 30. The compositionof claim 5, wherein X4 is iodated phenylalanine.
 31. The composition ofclaim 1 having the general formula:


32. A composition of somatostatin analogues having the general formula:

wherein: Z may be absent or present and when present is selected fromthe group consisting of DOTA-based chelators, DTPA-based chelators,NOTA-based chelators, carbonyl compounds, hydrazino nicotinamide,N₄-chelators, desferrioxamine, N_(X)S_(Y)-chelators, tyrosine forhalogenation, a fluorescent dye, and biotin; L may or may not be presentand is a linker molecule; and X4 is Phe or Tyr.
 33. The composition ofclaim 32, further comprising a radioactive isotope selected from thegroup consisting of ^(99m)Tc, ²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ¹¹¹In, ¹¹³In,¹²³I, ¹²⁴I, ¹⁸F, ¹⁷⁷Lu, ⁹⁷Ru, ⁶²Cu, ⁶⁴Cu, ⁵²Fe, ^(52m)Mn and ⁵¹Cr. 34.The composition of claim 32, further comprising a radioisotope selectedfrom the group consisting of ^(114m)In ¹⁸⁶Re, ¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁶Ga,⁶⁷Cu, ¹⁶⁹Er, ^(117m)Sn, ¹²¹Sn, ¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁴⁹Tb,¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴⁹Pm, ¹⁵¹Pm, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷²Tm,¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh, ¹¹¹Ag, ¹²⁴I, and ¹³¹I.